The present invention relates to low temperature expression cDNAs encoding fructan synthesizing enzymes and a method of isolating the same. In detail, this invention relates to cDNAs encoding enzymes effective for synthesizing a fructan which is useful for improving cold resistance of plants and can serve as a fructo-oligosaccharide useful for improving human""s health. In particular, this invention relates to the provision of a novel sucrose:fructan 6-fructosyltransferase cDNA isolated from a winter wheat (PI173438) having high freezing tolerance and a novel invertase cDNA isolated from the same winter wheat (PI173438).
It has been known that wintering crops such as wheats and forage grasses will acquire a freezing tolerance induced during a cold acclimation from autumn to winter. Further, it has been found that these wintering crops have fructan synthesizing enzymes which will express under a low temperature during cold acclimation, and as a result, polysaccharide fructan will be synthesized and accumulated as an energy source within vacuoles of these crops. Since wintering crops are often planted in severely cold areas, it is desired that the fructan be accumulated as large amount as possible.
Moreover, it has been found that the fructan can serve not only as an energy source for a plant to live through a severe winter, but also can serve to prevent a plant from freezing and to adjust the osmotic pressure of a plant so as to prevent it from desiccation.
Recently, some new wheat varieties have been developed in some northern countries, but it has been proved difficult to obtain a new wheat variety having more improved capabilities than those of existing ones, if we only use a conventional hybridization technique. For this reason, there has been a demand that research and development of a new wheat variety be conducted by means of gene engineering.
On the other hand, fructan can serve as a fructo-oligosaccharide which has been proved useful for preventing a carious tooth and for increasing intestinal bifid bacteria (useful for improving human health). In fact, a cDNA encoding fructan synthesizing enzyme (it may also be referred to as fructan synthesizing cDNA) is useful for producing the fructo-oligosaccharide at an industrial level.
In detail, enzymes capable of synthesizing fructan may be classified into several species and can have several isozymes in view of different reaction stages and different combined states. In more detail, sucrose:fructan 6-fructosyltransferase is mainly useful for synthesizing a trisaccharide or a tetraccharide using sucrose as starting substance and for extending a degree of fructose polymer using fructan. Invertase can usually serve as an enzyme for decomposing the sucrose (disaccharide). Further, invertase of plants possessing a fructan accumulation ability (such plants include wheat and forage grasses) may be used to synthesize a trisaccharide in the presence of a high concentration sucrose.
It is reported that the sucrose:fructan 6-fructosyltransferase cDNA may be isolated from barley, while the invertase cDNA may be isolated from several other kinds of plants. However, since these cDNAs are all isolated under a condition suitable for cDNAs to express at the room temperature, it is not sure whether they can express at a low temperature and hence they may not have a function as an enzyme under a low temperature environment. As a result, the conventional fructan 6-fructosyltransferase cDNA and the conventional invertase cDNA can not be used to improve the cold resistance of a plant.
It is an object of the present invention to provide cDNAs encoding fructan synthesizing enzymes capable of expressing under a low temperature environment, which cDNAs are isolated from a winter wheat (PI173438) having a high freezing tolerance.
It is another object of the present invention to provide cDNAs encoding fructan synthesizing enzymes useful for industrially producing fructo-oligosaccharides which are useful for preventing a carious tooth and for increasing intestinal bifid bacteria.
It is a further object of the present invention to provide a method for isolating the above cDNAs capable of expressing in a low temperature environment.
According to one aspect of the present invention, there is provided a wheat-derived sucrose:fructan 6-fructosyltransferase cDNA, characterized in that said cDNA has a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.1 in FIG. 1. In detail, said cDNA comprises 1848 nucleotides/616 amino acids and has 93% identity (on amino acid sequence level) with barley-derived sucrose:fructan 6-fructosyltransferase cDNA. In more detail, said cDNA encodes a fructan synthesizing enzyme in a low temperature environment and can confer a cold resistance to a plant upon being introduced into the plant. In further detail, said cDNA encodes an enzyme having a function of synthesizing a fructo-oligosaccharide useful for improving human""s health.
According to one aspect of the present invention, there is provided a wheat-derived invertase cDNA, characterized in that said cDNA has a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.2 in FIG. 2. In detail, said cDNA comprises 1986 nucleotides/662 amino acids and has 55% identity (on amino acid sequence level) with sugarcane-derived invertase cDNA. In more detail, said cDNA encodes a fructan synthesizing enzyme in a low temperature environment and can confer a cold resistance to a plant upon being introduced into the plant. In further detail, said cDNA encodes an enzyme having a function of synthesizing a fructo-oligosaccharide useful for improving human""s health.
According to a further aspect of the present invention, there is provided a method of isolating a wheat-derived sucrose:fructan 6-fructosyltransferase cDNA having a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.1 in FIG. 1 and a wheat-derived invertase cDNA having a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.2 in FIG. 2, said method comprising the steps of: extracting mRNA from winter wheat variety PI173438 (having a high freezing tolerance) that has undergone a sufficient cold acclimation; preparing cDNA and a cDNA library based on said mRNA; analyzing nucleotide sequences of a number of plant-derived sucrose:fructan 6-fructosyltransferase cDNAs and plant-derived invertase cDNAs which have all been published by EMBL/Genebank/DDBJDNA Databank; designing a pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers with reference to highly conserved nucleotide sequence portions of the sucrose:fructan 6-fructosyltransferase cDNAs and the invertase cDNAs; conducting PCR (polymerase chain reaction) using the pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers and using said cDNA as a template, thereby amplifying fragments of sucrose:fructan 6-fructosyltransferase cDNA and invertase cDNA and obtaining amplified DNA fragments; and using said amplified DNA fragments as probes for screening said cDNA library by a hybridization assay, to isolate recombinant plaques containing full length of cDNA.
In the method of the present invention, one of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers has the following nucleotide sequence:
(Forward): 5xe2x80x2 A-T-G-A-A-T/C-G-A-T/C-C-C-N-A-A-T/C-G-G (SEQ.ID. No.3)
and the other has the following nucleotide sequence:
(Reverse): 5xe2x80x2 C-C-N-G-T-N-G-C-A/G-T-T-A/G-T-T-A-/G-A-A (SEQ.ID. No.4).
The above objects and features of the present invention will become better understood from the following description with reference to the accompanying drawings.